The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK

Nischarin, a novel intracellular protein, was originally identified as a binding partner for the alpha5beta1 integrin. Here we show that Nischarin also interacts with members of the PAK family of kinases. The amino terminus of Nischarin preferentially binds to the carboxy-terminal domain of PAK1 when the kinase is in its activated conformation. Nischarin binding to PAK1 is enhanced by active Rac, with the three proteins forming a complex, while expression of the alpha5beta1 integrin also increases the Nischarin/PAK1 association. Interaction with Nischarin strongly inhibits the ability of PAK1 to phosphorylate substrates. This effect on PAK kinase activity closely parallels Nischarin's ability to inhibit cell migration. Conversely, reduction of endogenous levels of Nischarin by RNA interference promotes cell migration. In addition, PAK1 and Nischarin colocalize in membrane ruffles, structures known to be involved in cell motility. Thus, Nischarin may regulate cell migration by forming inhibitory complexes with PAK family kinases.     

NMR binding and crystal structure reveal that intrinsically-unstructured regulatory domain auto-inhibits PAK4 by a mechanism different from that of PAK1

Six human PAK members are classified into groups I (PAKs 1–3) and II (PAK4–6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs.     

Substrate and Inhibitor Specificity of the Type II p21-Activated Kinase,PAK6

The p21-activated kinases (PAKs) are important effectors of Rho-family small GTPases. The PAK family consists of two groups, type I and type II, which have different modes of regulation and signaling. PAK6, a type II PAK, influences behavior and locomotor function in mice and has an ascribed role in androgen receptor signaling. Here we show that PAK6 has a peptide substrate specificity very similar to the other type II PAKs, PAK4 and PAK5 (PAK7). We find that PAK6 catalytic activity is inhibited by a peptide corresponding to its N-terminal pseudosubstrate. Introduction of a melanoma-associated mutation, P52L, into this peptide reduces pseudosubstrate autoinhibition of PAK6, and increases phosphorylation of its substrate PACSIN1 (Syndapin I) in cells. Finally we determine two co-crystal structures of PAK6 catalytic domain in complex with ATP-competitive inhibitors. We determined the 1.4 Å co-crystal structure of PAK6 with the type II PAK inhibitor PF-3758309, and the 1.95 Å co-crystal structure of PAK6 with sunitinib. These findings provide new insights into the structure-function relationships of PAK6 and may facilitate development of PAK6 targeted therapies.     

Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines

p21-activated protein kinase (PAK) serine/threonine kinases are important effectors of Rho family GTPases and have been implicated in the regulation of cell morphology and motility, as well as in cell transformation. To further investigate the possible involvement of PAK kinases in tumorigenesis, we analyzed the expression of several family members in tumor cell lines. Here we demonstrate that PAK4 is frequently overexpressed in human tumor cell lines of various tissue origins. We also have identified serine (Ser-474) as the likely autophosphorylation site in the kinase domain of PAK4 in vivo. Mutation of this serine to glutamic acid (S474E) results in constitutive activation of the kinase. Phosphospecific antibodies directed against serine 474 detect activated PAK4 on the Golgi membrane when PAK4 is co-expressed with activated Cdc42. Furthermore, expression of the active PAK4 (S474E) mutant has transforming potential, leading to anchorage-independent growth of NIH3T3 cells. A kinase-inactive PAK4 (K350A,K351A), on the other hand, efficiently blocks transformation by activated Ras and inhibits anchorage-independent growth of HCT116 colon cancer cells. Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth.     

The active conformation of the PAK1 kinase domain

The p21-activated kinases (PAKs) participate in cytoskeletal control networks, downstream of Rho-family GTPases. A structure of PAK1 in an autoregulated, "off" state showed that a regulatory region, N-terminal to the kinase domain, forces the latter into an inactive conformation, prevents phosphorylation of Thr423 in the activation loop, and promotes dimerization. We have now determined structures at 1.8 A resolution for the free PAK1 kinase domain, with a mutation in the active site that blocks enzymatic activity, and for the same domain with a "phosphomimetic" mutation in the activation loop. The two very similar structures show that even in the absence of a phosphorylated Thr423, the kinase has an essentially active conformation. When Cdc42 binds the regulatory region and dissociates the dimer, PAK1 will be in an "intermediate-active" state, with a capacity to phosphorylate itself or other substrates even prior to modification of its activation loop.     

Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).     

Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.     

PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.

We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.     

The structure of MSK1 reveals a novel autoinhibitory conformation for a dual kinase protein

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.     

PAK5, a New Brain-Specific Kinase, Promotes Neurite Outgrowth in N1E-115 Cells

We have characterized a new member of the mammalian PAK family of serine/threonine kinases, PAK5, which is a novel target of the Rho GTPases Cdc42 and Rac. The kinase domain and GTPase-binding domain (GBD) of PAK5 are most closely related in sequence to those of mammalian PAK4. Outside of these domains, however, PAK5 is completely different in sequence from any known mammalian proteins. PAK5 does share considerable sequence homology with the Drosophila MBT protein (for "mushroom body tiny"), however, which is thought to play a role in development of cells in Drosophila brain. Interestingly, PAK5 is highly expressed in mammalian brain and is not expressed in most other tissues. We have found that PAK5, like Cdc42, promotes the induction of filopodia. In N1E-115 neuroblastoma cells, expression of PAK5 also triggered the induction of neurite-like processes, and a dominant-negative PAK5 mutant inhibited neurite outgrowth. Expression of activated PAK1 caused no noticeable changes in these cells. An activated mutant of PAK5 had an even more dramatic effect than wild-type PAK5, indicating that the morphologic changes induced by PAK5 are directly related to its kinase activity. Although PAK5 activates the JNK pathway, dominant-negative JNK did not inhibit neurite outgrowth. In contrast, the induction of neurites by PAK5 was abolished by expression of activated RhoA. Previous work has shown that Cdc42 and Rac promote neurite outgrowth by a pathway that is antagonistic to Rho. Our results suggest, therefore, that PAK5 operates downstream to Cdc42 and Rac and antagonizes Rho in the pathway, leading to neurite development.     

Cdc42/Rac1-mediated activation primes PAK2 for superactivation by tyrosine phosphorylation

The involvement of p21-activated kinases (PAKs) in important cellular processes such as regulation of the actin skeleton morphology, transduction of signals controlling gene expression, and execution of programmed cell death has directed attention to the regulation of the activity of these kinases. Here we report that activation of PAK2 by p21 GTPases can be strongly potentiated by cellular tyrosine kinases. PAK2 became tyrosine phosphorylated in its N-terminal regulatory domain, where Y130 was identified as the major phosphoacceptor site. Tyrosine phosphorylation-mediated superactivation of PAK2 could be induced by overexpression of different Src kinases or by inhibiting cellular tyrosine phosphatases with pervanadate and could be blocked by the Src kinase inhibitor PP1 or by mutating the Y130 residue. Analysis of PAK2 mutants activated by amino acid changes in the autoinhibitory domain or the catalytic domain indicated that GTPase-induced conformational changes, rather than catalytic activation per se, rendered PAK2 a target for tyrosine phosphorylation. Thus, PAK activation represents a potentially important point of convergence of tyrosine kinase- and p21 GTPase-dependent signaling pathways.     

Targeted disruption of the gene for the PAK5 kinase in mice

PAK5 is a member of the group B family of PAK serine/threonine kinases and is an effector for the Rho GTPase Cdc42. PAK5 is highly expressed in the brain and is expressed at lower levels in several other tissues. In cell lines, PAK5 has been shown to play a role in filopodia formation and neurite outgrowth. To examine the biological function of PAK5, we deleted the PAK5 gene in mice. The phenotypes of the PAK5-null mice are completely different from those of mice null for PAK4, another member of the group B PAK family. Unlike PAK4-null mice, which are embryonic lethal, PAK5-null mice develop normally and are fertile. The nervous system appears normal in the absence of PAK5, as do other tissues in which PAK5 is normally expressed. Our results suggest functional redundancy between PAK5 and other Rho GTPase targets.     

Group I and II mammalian PAKs have different modes of activation by Cdc42

p21‐activated kinases (PAKs) are Cdc42 effectors found in metazoans, fungi and protozoa. They are subdivided into PAK1‐like (group I) or PAK4‐like (group II) kinases. Human PAK4 is widely expressed and its regulatory mechanism is unknown. We show that PAK4 is strongly inhibited by a newly identified auto‐inhibitory domain (AID) formed by amino acids 20 to 68, which is evolutionarily related to that of other PAKs. In contrast to group I kinases, PAK4 is constitutively phosphorylated on Ser 474 in the activation loop, but held in an inactive state until Cdc42 binding. Thus, group II PAKs are regulated through conformational changes in the AID rather than A‐loop phosphorylation.     

Adhesion stimulates direct PAK1/ERK2 association and leads to ERK-dependent PAK1 Thr212 phosphorylation

The Rac1/Cdc42 effector p21-activated kinase (PAK) is activated by various signaling cascades including receptor-tyrosine kinases and integrins and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical Ras/Raf/MEK/ERK Map kinase signaling cascade, likely because of PAK co-activation of Raf and MEK. Herein, we found that adhesion signaling also stimulates an association between PAK1 and ERK1/2. PAK1 and ERK1/2 co-immunoprecipitated from rat aortic smooth muscle cells (SMC) plated on fibronectin, and the two proteins co-localized in membrane ruffles and adhesion complexes following PDGF-BB or sphingosine 1-phosphate treatment, respectively. Far Western analysis demonstrated a direct association between the two proteins, and peptide mapping identified an ERK2 binding site within the autoinhibitory domain of PAK1. Interestingly, deletion of a major ERK binding site in PAK attenuates activation of an ERK-dependent serum-responsive element (SRE)-luciferase reporter gene, indicating that association between PAK and ERK is required to facilitate ERK signaling. We also show that ERK2 phosphorylates PAK1 on Thr(212) in vitro and that Thr(212) is phosphorylated in smooth muscle cells following PDGF-BB treatment in an adhesion- and MEK/ERK-dependent fashion. Expression of a phosphomimic variant, PAK-T212E, does not alter ERK association, but markedly attenuates downstream ERK signaling. Taken together, these data suggest that PAK1 may facilitate ERK signaling by serving as a scaffold to recruit Raf, MEK, and ERK to adhesion complexes, and that subsequent growth factor-stimulated phosphorylation of PAK-Thr(212) by ERK may serve to provide a negative feedback signal to control coordinate activation of ERK by growth factor- and matrix-induced signals.     

Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin.

PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in response to the Rho GTPase Cdc42. Here we show that PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 was shown to interact specifically with LIMK1 in binding assays. Immune complex kinase assays revealed that both wild-type and constitutively active PAK4 phosphorylated LIMK1 even more strongly than PAK1, and activated PAK4 stimulated LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments revealed that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibited the specific cytoskeletal and cell shape changes that were induced in response to a recently characterized constitutively activated PAK4 mutant.     

p21-activated kinase PAK phosphorylates desmin at sites different from those for Rho-associated kinase

p21-activated kinase (PAK) and Rho-associated kinase (Rho-kinase) have been shown to induce Ca(2+)-independent contraction of smooth muscle. PAK-induced contraction of Triton-skinned smooth muscle correlates with increased phosphorylation of caldesmon and desmin, although the role of desmin phosphorylation has remained obscure. Here we report that desmin serves as an excellent substrate for PAK in vitro. PAK phosphorylated desmin in a GTP. Cdc42/Rac-dependent manner. Phosphorylation of desmin by PAK dramatically inhibited its filament-forming ability. PAK phosphorylated mainly serine residues of the head domain of desmin, and the major phosphorylation sites differed from those for Rho-kinase. These results suggest that different site-specific phosphorylation of desmin via two divergent protein kinases downstream of Rho family GTPases would seem to increase the regulatory potential for organization of desmin filaments.     

Activation of myosin in HeLa cells causes redistribution of focal adhesions and F-actin from cell center to cell periphery

Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells.